Bacterial biofilms have been directly implicated in the development of antibacterial resistance of clinical infections. Biofilms of Pseudomonas aeruginosa account for 10% of all nosocomial infections and are the leading cause of chronic infections in Cystic Fibrosis patients. Ga3+ salts have recently emerged as effective antibiotic agents against P. aeruginosa biofilms, however little is known about their mechanism of action. The identification of the P. aeruginosa proteome in response to antibiofilm agents would give significant insight into the mechanism of antibiotic resistance;however, there are limited technologies to identify newly synthesized proteins. Bioorthogonal non-canonical amino acid tagging (BONCAT) has been successful in identifying newly synthesized proteins with high resolution. By metabolically labeling proteins with amino acids bearing a chemical reporter, only proteins with the new amino acid will be identified in post-translational affinity tagging. While powerful, successful incorporation of an artificial amino acid is highly dependent on the cellular translational machinery. To address this issue we propose using L-selenomethionine (SeMet) as a surrogate amino acid for protein labeling due to its near complete incorporation into proteins using endogynous translational machinery and minimal effect on protein function. Post-translational oxidation and elimination of SeMet residues to form alkenes can be coupled with thiol-ene chemistry to tag newly synthesized proteins, eliminating the need for a chemical reporter. This project's long-term objective is to develop a SeMet-BONCAT method to identify low-abundant proteins essential to understand time-dependent proteome changes in biofilms. To achieve this goal, we propose the following aims: (1) develop a methodology to promote alkene formation from oxidized SeMet residues in aqueous media, (2) metabolically label proteins with SeMet and selectively tag newly synthesized proteins using thiol-ene chemistry in Pseudomonas aeruginosa Met- auxotrophs, and (3) identify new proteins from Pseudomonas aeruginosa biofilms in response to treatment with Ga3+ salts exposure. Experiments will involve using Lewis acid surfactant-combined catalysts (LASCs) to promote alkene formation from SeMet residues and tandem 2D chromography and mass spectrometry for protein identification. We anticipate that this new protein labeling and tagging strategy will provide significant insight and understanding into the biological processes and molecular mechanisms of biofilms and other organisms of clinical relevance. PUBLIC HEALTH RELEVANCE: The relevance of the proposed research to public health is centered upon understanding the molecular mechanisms of antibiotic resistance in bacterial biofilms. By understanding these mechanisms, effective treatments can be developed to combat antibiotic resistant infections in clinical settings.